61Cu-PSMA–Targeted PET for Prostate Cancer: From Radiotracer Development to First-in-Human Imaging

Visual Abstract

PET that targets prostate-specific membrane antigen (PSMA) continues to grow in usage.It has shown clinical value in the initial staging of newly diagnosed high-risk prostate cancer, localization of disease sites in patients with biochemical recurrence, and identification of appropriate patients for PSMA-targeted radiopharmaceutical therapy (1)(2)(3)(4).Several PSMA-targeted PET tracers have been developed, with most bearing Glu-urea-Lys as the binding motif (5,6).Among them, [ 68 Ga]Ga-PSMA-11 ( 68 Ga-gozetotide), [ 18 F]DCFPyL ( 18 F-piflufolastat), and [ 18 F]rhPSMA-7.3( 18 F-flotufolastat) are approved by the Food and Drug Administration.With PSMA-targeted radiopharmaceutical therapy becoming an important option (7,8), the demand for PSMA PET scans is expected to expand rapidly across the globe (9).
So far, only 68 Ga-and 18 F-labeled PSMA tracers have been used (5,6).However, the relatively short half-life (t 1/2 ) of 68 Ga (t 1/2 , 68 min) and of 18 F (t 1/2 , 110 min) limits the typical geographic distribution range of these radiotracers to about 160 km (100 miles).Because not all medical centers have radiochemistry facilities or exist close to radiotracer production sites, there are substantial gaps in geographic coverage for these PET tracers.

61
Cu has the following advantages as a radioisotope for PET imaging.First, 61 Cu can be produced in cyclotrons on a large scale, similar to 18 F, and combines the attractive logistics of centralized radiotracer production with chelator-based radiochemistry, similar to 68 Ga (10).Transport by land to remote PET facilities is possible up to a radius of 300 km (186 miles) within 1 t 1/2 .Fewer than 10 production sites would be required to supply 61 Cu or 61 Cu-labeled tracers to most populous regions in the continental United States or Europe (11,12).Second, the longer physical t 1/2 of 61 Cu enables delayed imaging when image contrast will be higher, because the radioactivity from PSMA ligands is cleared only slowly from the tumor cells.Third, 61 Cu can be paired with b 2 -emitting 67 Cu to create true theranostic twins for imaging and therapy agents.
Despite the advantages of 61 Cu, it has not been widely used for the development of PET tracers, mainly because of its lack of availability, low radionuclide purity, and low yields (10).These issues have been addressed recently (13,14).In 2020, Svedjehed et al. (14) developed an automated procedure for isolating [ 61 Cu]CuCl 2 from cyclotron-irradiated Ni targets.The increased availability of 61 Cu opens opportunities for its use.We report herein the development of the first 61 Cu-labeled PSMAtargeted tracers.We used PSMA-I&T (DOTAGA-(I-y)fk(sub-KuE)), which was evaluated in a phase 2 clinical trial labeled with 64 Cu (NCT05653856).In parallel, we developed the new NODAGA derivative of PSMA-I&T (NODAGA-(l-y)fk(sub-KuE)), based on our previous work demonstrating advantages of the chelator NODAGA over DOTA-or cyclam-based chelators for 64 Cu (15,16).The 2 derivatives, herein reported as DOTAGA-PSMA-I&T and NODAGA-PSMA-I&T, were used for 61 Cu radiotracer development, in vitro and in vivo characterization, and first-in-human imaging.

MATERIALS AND METHODS
All information on the reagents, analytic methods, cell line, and experimental procedures are provided in the supplemental materials (supplemental materials are available at http://jnm.snmjournals.org).

Production and Purification of [ 61 Cu]CuCl 2
[ 61 Cu]CuCl 2 was produced by irradiating natural nickel ( nat Ni) electroplated on silver coins at 40 mA over 120 min in a GE Healthcare medical cyclotron at the University Hospital Zurich, Switzerland, followed by purification based on Svedjehed et al. (14).The process yielded approximately 1 GBq/mL [ 61 Cu]CuCl 2 in 0.05 M HCl.Details on the production, purification, and extraction of 61 Cu will be published elsewhere.

Animal Studies
All animal experiments were conducted in accordance with Swiss animal welfare laws and regulations under license number 30515 granted by the Veterinary Office, Department of Health, Canton Basel-Stadt, Switzerland.Male athymic nude-Foxn1 nu /Foxn1 1 mice (Envigo), 4-6 wk old, were inoculated subcutaneously on the shoulder with 10 7 LNCaP cells, freshly suspended in a 1:1 ratio of sterile minimum essential medium with basal medium Eagle and Matrigel.The tumors were allowed to grow to a volume of approximately 200 mm 3 .

In Vivo Metabolic Stability
The stability of [ 61 Cu]Cu-DOTAGA-PSMA-I&T and [ 61 Cu]Cu-NODAGA-PSMA-I&T was assessed by radio-reversed-phase highperformance liquid chromatography in urine and in liver and kidney homogenates from healthy BALB/c mice after injection of 100 mL/400 pmol/8-9 MBq of each radiotracer.Details are provided in the supplemental materials.

Dosimetry
Additional biodistribution data were generated in healthy BALB/c mice using [ 64 Cu]Cu-NODAGA-PSMA-I&T at 1, 4, 12, and 24 h after injection and combined with the data of [61 Cu]Cu-NODAGA-PSMA-I&T at 1 and 4 h after injection.Non-decay-corrected biodistribution data for 61 Cu (t 1/2 , 3.33 h) were used to generate time-activity curves for [ 61 Cu]Cu-NODAGA-PSMA-I&T.OLINDA/EXM version 1.0 (Vanderbilt University) was used for the dosimetry estimates, as described in the supplemental materials.are described in the supplemental materials.A dose of 105 MBq/32 mg was administered intravenously to a patient with known metastatic prostate cancer before 177 Lu-labeled PSMA radiopharmaceutical therapy.The patient was coinjected with 10 mg of furosemide (Lasix; Sanofi-Aventis).Imaging was performed 3 h after tracer administration on a Biograph Vision PET/CT scanner (Siemens Healthineers).Images were obtained from the skull to mid-thigh and reconstructed into multiplanar PET, CT, and fused PET/CT images.CT was used for attenuation correction.

Statistics
Statistical analysis was performed by unpaired t testing with Welch correction using GraphPad Prism version 9 (GraphPad Software).P values of less than 0.05 were considered significant.All data were evaluated as mean 6 SD.

RESULTS
61 Cu-PSMA Tracers: Radiochemistry and In Vitro Characterization 61 Cu production by solid target irradiation of nat Ni, followed by purification as described earlier, resulted in a 1.4-to 2.1-GBq yield and more than 99.99% radionuclidic purity at 12 h after purification.Details on production and quality control results of the [ 61 Cu]CuCl 2 solution used for radiolabeling will be published elsewhere.The analytic data and the in vitro properties of [ 61 Cu]Cu-DOTAGA-PSMA-I&T and [ 61 Cu]Cu-NODAGA-PSMA-I&T are summarized in Table 1.

First-in-Human PET/CT Imaging
Administration of [ 61 Cu]Cu-NODAGA-PSMA-I&T (specifications are provided in Supplemental Table 7) and imaging were performed (Fig. 4).Radiotracer accumulation was noted in multifocal osseous and hepatic metastases, and the physiologic distribution of PSMA-targeted tracers was as expected in the lacrimal glands, salivary glands, liver, spleen, kidneys, ureters, bladder,  and proximal small bowel.The SUV max and SUV mean were as follows: salivary gland (right parotid gland), 20.5 and 12.6; liver, 6.5 and 3.4; and kidney (right), 57.3 and 37.9, respectively.For the tumor lesions, the SUV max and SUV mean were as follows: right lower pubic bone, 154.2 and 83.7; right scapula, 86.4 and 55.3; left liver lobe, 23.6 and 11.7; and sacral bone, 16.5 and 9.9, respectively.

DISCUSSION
PSMA-targeted PET imaging has become a new standard of care for patients with prostate cancer (4,17,18).This study aimed to assess the feasibility of 61 Cu-PSMA-targeted tracers in terms of performance, clinical relevance, ease of production, and accessibility.We aimed to provide insights into the advantages of this tracer compared with others in its class.In vivo, [ 61 Cu]Cu-NODAGA-PSMA-I&T showed clear superiority over [ 61 Cu]Cu-DOTAGA-PSMA-I&T by means of higher tumor uptake (P 5 0.0050 1 h after injection vs. P 5 0.0066 4 h after injection), lower blood-pool activity, and especially liver and abdominal activity.The metabolic instability of [ 61 Cu]Cu-DOTAGA-PSMA-I&T and the release of 61 Cu from the DOTAGA chelator led to activity accumulation in the liver.Similar findings were shown previously in vivo with [ 64 Cu]Cu-PSMA-617, in which DOTA was used as a chelator (19).Other 64 Cu-labeled PSMA ligands reported in the literature are using more suitable chelators for Cu(II) than DOTA, such as cyclams (20) or sarcophagine (21), and different Glu-urea-Lys motifs.Among them, the dimer [ 64 Cu]Cu-sarcophagine-bisPSMA is in a phase 3 trial (NCT06056830).To our knowledge, the conjugate NODAGA-PSMA-I&T under investigation has not been reported.Overall, our in vivo studies with [ 61 Cu]Cu-NODAGA-PSMA-I&T show biodistribution similar to that of [ 68 Ga]Ga-PSMA-11 and [ 18 F]PSMA-1007 at 1 h after injection.At the later time of 4 h after injection, [ 61 Cu]Cu-NODAGA-PSMA-I&T had improved tumorto-background ratios, demonstrating the advantage of using a longer-t 1/2 radionuclide to optimize radiotracer biodistribution and tumor-to-background contrast.Dosimetry estimates of [ 61 Cu]Cu-NODAGA-PSMA-I&T suggested that dosimetry is within the expected levels of the 68 Ga-labeled and 18 F-labeled PSMA tracers (22,23).Higher tumor-to-background ratios obtained in scans at later time points with [ 61 Cu]Cu-NODAGA-PSMA-I&T can potentially enhance the detection rate of lesions and provide clarification of findings that were unclear in scans at early time points.This observation is supported by several studies that have shown increased PSMA lesion detection rates when scans are performed beyond the initial 1-h window using various tracers, such as [ 68 Ga]Ga-PSMA (22,24,25), 18 F-labeled PSMA tracers (26), or more recently 99m Tc-labeled PSMA tracers (27).
This work takes the newly developed 61 Cu-PSMA-targeted tracer into successful first-in-human imaging.Although a single subject does not guarantee future success, we are highly encouraged by the prominent tracer uptake in both osseous and hepatic metastases, which are clearly visualized.
Most PSMA-targeted PET tracers use 68 Ga and 18 F for radiolabeling.Thus far, the implementation of these tracers has been constrained by the relatively short t 1/2 of 68 Ga and 18 F, which restricts the efficient distribution of tracers beyond a limited geographic range and the availability of delayed imaging.Centralized production facilities of 18 F-labeled tracers are generally confined to distribution areas of a few hundred miles, requiring substantial networks of production facilities.Even with multiple production sites, wide  areas of the population may not be able to receive and use these radionuclides.
Copper radioisotopes are attractive for use in both molecular imaging and therapy, because positron-emitting 61 Cu (t 1/2 , 3.33 h) and 64 Cu (t 1/2 , 12.7 h) agents may be paired with b 2 -emitting 67 Cu agents to create true theranostic pairs. 64Cu has been used previously in PET imaging tracers such as [ 64 Cu]Cu-DOTATATE, given its commercial availability (28). 64Cu has a longer t 1/2 (12.7 h) than 68 Ga and 18 F, allowing greater geographic distribution of products and delayed imaging.However, 64 Cu is limited by its low positron yield (18%), which may impair image quality, and 39% of its decays are b 2 -emissions, increasing radiation exposure.Compared with 64 Cu, 61 Cu combines the advantage of long t 1/2 with far greater positron yield (61%) yet lacks high-energy b 2 -emissions (29).The physical properties of 61 Cu versus 64 Cu are compared in Supplemental Table 8.
Despite the favorable physical properties of 61 Cu, the literature has reported only a few instances of ligands labeled with this radionuclide (30)(31)(32).This scarcity can be attributed primarily to the limited availability and distribution of 61 Cu.However, recent advances in the automated cyclotron production of [ 61 Cu]CuCl 2 using liquid zinc (13) and solid nickel targets (14) have paved the way for greater accessibility to 61 Cu and subsequently expanded its potential for clinical applications (10).
With inexpensive nat Ni as the starting material, highly pure 61 Cu could be produced with radionuclidic purity exceeding 99.99% at 12 h after synthesis (details on the production will be published elsewhere).The preparation of [ 61 Cu]Cu-NODAGA-PSMA-I&T was performed in widely used buffers at room temperature within 5 min.The radiolabeling process at the apparent molar activity of 24 MBq/nmol demonstrated high yield (.98%) and stability ($97% up to 4 h).Moreover, [ 61 Cu]Cu-NODAGA-PSMA-I&T synthesized in a good manufacturing practice grade for human use was found to be stable for up to 9 h at room temperature, at an activity concentration of 20 MBq/nmol (Supplemental Table 9).These results surpass the typical yields achieved with 18 F-labeled PSMA tracers in the same class (33).High labeling yields and suitable molar activities for clinical use eliminate the need for any purification step after labeling.Furthermore, the production of 61 Cu can be scaled up to meet the growing demand.61 Cu production necessitates 1-3 h of cyclotron beam time, and its yield varies from 3 to 100 GBq, depending on the enrichment of the starting nickel material ( 60 Ni and 61 Ni) and beam parameters.These are advantageous features compared with 64 Cu production, which demands 4-12 h of beam time for a 3-to 10-GBq yield and necessitates highly enriched (.98%) 64 Ni to achieve the necessary radionuclidic purity and specific activity.All of these factors play crucial roles in determining the ease of production, scalability, and overall viability of the tracer for practical implementation in clinical settings.

CONCLUSION
This study demonstrates the successful development, in vitro and in vivo characterization, and first-in-human imaging of 61 Cu-labeled tracers for PSMA targeting.[ 61 Cu]Cu-NODAGA-PSMA-I&T had better biodistribution, pharmacokinetics, and imaging properties than [ 61 Cu]Cu-DOTAGA-PSMA-I&T.It also compared favorably with [ 68 Ga]Ga-PSMA-11 and [ 18 F]PSMA-1007 and demonstrated advantages at delayed imaging times.The study highlights the straightforward production of a high-quality 61 Cu-labeled PSMAtargeted tracer suitable for future implementation.Several factors, such as radiochemical yield, radiochemical purity, and stability, that significantly affect a PET tracer's production, distribution, and clinical viability were also assessed.Imaging with [ 61 Cu]Cu-NODAGA-PSMA-I&T successfully visualized multifocal metastatic prostate cancer.Overall, the findings of this study serve as a foundation for future clinical development of 61 Cu-labeled tracers and suggest opportunities for development of other 61 Cu-labeled tracers for a range of clinically valuable targets.

DISCLOSURE
The study was financially support by the Swiss Innovation Agency (Innosuisse), project 37014.1 IP-LS, and by Nuclidium AG (matching funding).Melpomeni Fani reports research funding from Ipsen, ITM, and Nuclidium; acts as a scientific advisor of Nuclidium; and is coinventor on patent applications filed by Nuclidium and the University of Basel related to 61 Cu-labeled tracers.Francesco De Rose and Leila Jaafar-Thiel are employees of Nuclidium and coinventors in a series of patents related to 61 Cu-labeled tracers.Gary Ulaner discloses that he receives speaker fees and research support from Lantheus, GE Healthcare, and RayzeBio and serves on the scientific advisory boards of Lantheus, GE Healthcare, RayzeBio, and Nuclidium.Matthias Eiber reports fees from Blue Earth Diagnostics Ltd. (consultant and research funding), Novartis/AAA (consultant and speaker), Telix (consultant), Bayer (consultant and research funding), RayzeBio (consultant), Point Biopharma (consultant), Eckert-Ziegler (speaker), Janssen Pharmaceuticals (consultant and speakers bureau), Parexel (image review), and Bioclinica (image review) outside the submitted work.He and other inventors are entitled to royalties on sales of Posluma.Wolfgang Weber reports fees from Nuclidium, TRIMT, BMS, Ipsen, Imaginab, and Piramal (grants); RayzeBio, Bayer, Blue Earth Diagnostics, Pentixapharm, and Vida Ventures (consultant); GSK and AAA (speakers' bureau); and ITM, Endocyte, and Reflexion (advisory board).No other potential conflict of interest relevant to this article was reported.

TABLE 4
Total Absorbed Doses in Different Organs of [ 61 Cu]Cu-NODAGA-PSMA-I&T Calculated by OLINDA/EXM Version 1.0, with Assumption That Kinetics in Mouse Is Same as Kinetics in Human